Immunogenic glycopeptides, screening, preparation and uses

ABSTRACT

The invention concerns immunogenic glycopeptides derived from pathogenic microorganisms, useful for vaccination and diagnosis of infections caused by said pathogenic microorganisms (bacteria or fungi), and methods for selecting them and preparing them. Said glycopeptides are selected in the group consisting of: a1) glycopeptides essentially consisting of a glycosylated T epitope, comprising 14 to 25 amino acids, among which at least a neutral amino acid is bound to a di- or to a trisaccharide (glycoside linkage) and at least 15% among said amino acids are proline, one of the proline being located in position −1 to −4, relative to the position of said neutral amino acid, which glycopeptides are: exhibited by a class II MHC molecule, specifically identified by T CD+4 lymphocytes induced by immunisation with the native glycopeptide from which they are derived, but are not identified by the T CD+4 lymphocytes induced by immunisation with a non-glycosylated peptide of same sequence and capable of inducing a proliferation of said T CD+4 lymphocytes by which they are identified and the secretion of cytokines by said lymphocytes and b1) glycopeptides having a sequence of 15 to 39 amino acids including the sequence of the glycopeptide as defined in a1), excluding the glycopeptide of sequence SEQ ID NO:11.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 371 application of PCT/FR01/04100 filedDec. 20. 2001.

The present invention relates to immunogenic glycopeptides derived frompathogenic microorganisms, which can be used for immunization anddiagnosing infections due to such pathogenic microorganisms (bacteria orfungi), and also to the methods for the selection and for thepreparation thereof.

The means implemented for preventing and treating these infectionscomprise, firstly, screening which enables the infection to be monitoredand treated and, secondly, immunization.

These means are illustrated hereinafter, taking as an example one of themost serious infections in human medicine: infection with M.tuberculosis. Specifically, 5 to 10% of individuals infected with M.tuberculosis who have a normal immune response develop a serious disease(tuberculosis); this frequency is even higher in individuals who have adeficiency in their immune response (infection with HIV, treatment withimmuno-suppressors, etc.).

Diagnosis

Among the various techniques currently available, mention may be madeof:

-   -   the production of pure cultures of M. tuberculosis, which is the        most rigorous means for diagnosing tuberculosis with certitude.        It is a moderately sensitive technique which enables diagnosis        for ⅔ of the cases of pulmonary tuberculosis. The results are        available only after a minimum delay of 3-4 weeks, sometimes        only after culturing for 2 months. The use of culturing        techniques employing labelled precursors makes it possible to        shorten these delays, which nevertheless remain considerable.        This detection of M. tuberculosis by culturing requires a sample        containing bacilli, which is sometimes difficult to obtain even        for pulmonary tuberculosis, in which approximately ⅓ of cases do        not receive biological confirmation. Sometimes, this examination        requires a specialized medical intervention (lumbar puncture of        the cerebrospinal fluid or lymph node biopsy) for extrapulmonary        forms of the disease.    -   microbiological techniques based on molecular genetics (PCR) are        confronted with the same requirement of obtaining a sample        containing bacteria. Moreover, because of the presence, in the        sample, of PCR reaction inhibitors, the origin of which is        impossible to control, these techniques are sometimes unusable.        They have not been validated in common practice.    -   at the current time, there is no serodiagnosis which has a        sensitivity and a specificity compatible with diagnostic use.    -   the reaction to tuberculin shows that an individual is        sensitized, has been infected with M. tuberculosis or has been        immunized with BCG. Tuberculin is, in fact, a mixture of M.        tuberculosis antigens and is therefore incapable of making a        distinction between an infection with M. tuberculosis and        immunization with BCG, because of the very many cross-reactions        between the antigens of the vaccine and M. tuberculosis. In        addition, this reaction to tuberculin does not make it possible        to distinguish a tuberculosis, which is an active disease, from        an infection with M. tuberculosis.        Vaccine

Immunization with BCG makes it possible to control the primary infection(initial multiplication of M. tuberculosis) but especially the secondarydissemination of these bacilli. It probably contributes to decreasingthe incidence of latent infections against which no effective treatmentis currently available. BCG has been used to immunize more than 3billion individuals against tuberculosis, without any particular sideeffects. During immunization with BCG, there is a local multiplicationof these bacilli, of attenuated virulence. Cellular immunity is induced.It causes delayed-type hypersensitivity (HSR) directed against theproteins or antigens of mycobacteria (reaction to tuberculin), andincreased resistance to infection with M. tuberculosis. These two immuneresponses (HSR-type sensitization and increased resistance) aresupported by T lymphocytes reacting with mycobacterial antigens.

BCG protects well against the acute forms of the infection (tubercularmeningitis in children, for example). Its effectiveness is more variablein adults. The existence of a cross-reactivity between BCG and othermycobacteria which do not belong to the tuberculosis complex, and alsothe absence, in the BCG genome, of certain immunogenic antigens ofMycobacterium tuberculosis, or a different expression profile for theseantigens during the infection, may explain the variable effectiveness ofBCG.

In addition, BCG is a live strain of attenuated virulence. It thereforehas a residual pathogenic power which prohibits the use thereof inimmunodepressed individuals, in particular in individuals acknowledgedto be infected with the human immunodeficiency virus (HIV).

In order to combat these infections more effectively, it would bejudicious to have diagnostic tools and vaccines, in particular a“subunit” vaccine which therefore poses no danger, based on antigenswhich protect against the pathogenic microorganisms responsible forthese infections.

A certain number of studies have been carried out in this sense, inorder to find the molecule(s) of these pathogenic microorganisms, whichis (are) capable of inducing a strong protective immune response. Thus,J. Hess et al. (C. R. Acad. Sci. Paris, 1999, 322: 953-958) havereviewed the properties which antigens able to be used as a vaccineagainst tuberculosis should have. In that review, they underline theimportance of using a combination of preselected antigens rather than asingle antigen. They recommend, in particular, selecting these antigenson the basis of criteria such as the presence of regions which arehighly conserved among the various strains, the differences in the geneexpression profile of the virulent strains and of the attenuatedstrains, the reactivity with respect to the effector cells of the immuneresponse (B, CD4+ T, CD8+ T lymphocytes) or the capacity of theseantigens to bind to the majority of HLA molecules of the majorhistocompatibility complex (MHC).

Some of these antigens are present either in the form of surfaceantigens, such as the mannoproteins of C. albicans (Buurman et al.,PNAS, 1998, 95, 7670-7675), or in the form of secreted antigens, in M.tuberculosis: MPT59 (30 kDa), 85A (32 kDa), MPT64 (23 kDa), hsp71 (71kfla), MPT51 (24 kDa), MPT63 (16 kDa) and ESAT-6 (6 kDa), (Andersen,Infect. Immun., 1994, 62, 2536-2544; Horwitz et al., PNAS, 1995, 92,1530-1534). These M. tuberculosis antigens have already been proposed aspotential candidates for an immunization composition since they arepreferentially recognized by CD4+ T lymphocytes (Andersen, et al.,mentioned above; Horwitz et al., mentioned above).

It has also been proposed to isolate, from the M. tuberculosis antigens,peptides containing epitopes capable of being presented by an MHC classII molecule and of being recognized by specific CD4+ T lymphocytes; suchepitopes have in particular been reported for two proteins: ESAT-6(Olsen et al., Eur. J. Immunol., 2000, 30, 1724-1732) and MPT-39(Mustafa et al., Inf. Immunol., 2000, 68, 3933-3940).

Several observations have previously been made by the inventors (Romainet al., Inf. Immun., 1993, 61, 742-750; Romain et al. Proc. Natl. Acad.Sci. USA 1993, 90: 5322-5326):

-   -   only live bacteria are capable of inducing protective immunity,        killed bacteria also inducing an immune response, but without        protection;    -   in the culture medium, proteins exist which are released by the        bacteria, during their growth and which are capable of being        recognized by the immune system of animals immunized with live        bacteria, these being proteins which are poorly recognized or        not at all after immunization with killed bacteria.

Using this double criterion of selection, two new proteins have beenpurified. A protein secreted by M. tuberculosis, named Apa, or MPT-32 or45/47 kDa antigen complex, is the product of the Rv 1860 gene(Laqueyrerie et al. Infect. Immun. 1995, 63: 4003-4010). The secondmolecule is an internal peptide of a putative serine protease encoded bythe Rv 1796 gene.

In using the native Apa protein as an antigen, the inventors havepreviously shown that this protein, which represents only 2% of theproteins secreted by the bacilli of the tuberculosis group (M.tuberculosis, M. bovis and BCG) in culture, is an immunodominant antigenwhich is very effectively recognized by specific CD4+ T lymphocytesoriginating from animals infected with M. tuberculosis or immunized withBCG (Romain et al., Inf. Immun., 1999, 67, 5567-5572; Horn et al., J.Biol. Chem., 1999, 274, 32023-32030).

In these same studies, the inventors also showed that mannosylation ofApa was essential for the antigenic activity of this protein:

-   -   demannosylation of Apa, obtained by treating native Apa with        α-mannosidase or with trifluoromethane-sulphonic acid (TFMS), or        by expressing Apa in a bacterium incapable of glycosylating (E.        coli) is accompanied by a 100-fold loss of antigenicity,    -   glycosylated Apa produced by Mycobacterium smegmatis, which has        an overall mannose composition which is slightly different from        that of the Apa produced by M. tuberculosis, has an antigenic        activity which is decreased approximately 10-fold.

Moreover, it has been reported that this M. tuberculosis Apa moleculecontains 6 to 9 mannose residues linked, via a glycosidic bond of theα-(1,2) type, to 4 threonine residues (T₁₀, T₁₈, T₂₇ and T₂₇₇) in thefollowing way: a dimannose (T₁₀ and T₁₈), a mannose (T₂₇), a mannose, adimannose or a trimannose (T₂₇₇), (Dobos et al., J. Bacteriol., 1996,178, 2498-2506). It should be noted that this saccharide structure whichcontains mono-, di- or trimannoses resembles that of mannoproteins fromyeast, in particular from Candida albicans, and is different from thatof proteins from F. meningosepticum, which have longer oligomannosechains.

The loss of Apa antigenicity, observed after demannosylation, may be dueto a decrease in the phagocytosis and processing of this antigen, oralternatively in the recognition of the latter by CD4+ T lymphocytes.Specifically, the mannose receptor of macrophages and of dendriticcells, which bind specifically to hexoses, in particular ofmannoproteins from C. albicans and of mannolipids such aslipoarabinomannan from mycobacteria, plays a role in the phagocytosisand processing of antigens which are present at the surface of thesecells in the form of a peptide/class II MHC molecule complex (Stahl etal., Current Opinion in Immunology, 1998, 10, 50-55). It has also beenshown that a mannosylated peptide (mannosylated on lysine residues inthe N-terminal position) is phagocytosed and processed by dendriticcells much more effectively than a non-glycosylated peptide with thesame sequence (Tan et al., Eur. J. Immunol., 1997, 27, 2426-2435).

In the chicken lysozyme model, it has been shown that peptides which areglycosylated analogues of a peptide constituting a T epitope of thisantigen are capable of inducing CD4+ T lymphocytes which specificallyrecognize this glycosylated epitope (Deck et al., J. Immunol., 1995,155, 1074-1078). However, since such glycosylated T epitopesspecifically recognized by CD4+ T lymphocytes have not been identifiedin native antigens derived from pathogenic microorganisms(bacterium/fungus), the importance of glycosylation in the recognitionof antigens from these pathogenic microorganisms by CD4+ T lymphocytesremains to be demonstrated.

In addition, and this being despite the data relating to M. tuberculosisApa and general knowledge regarding the glycosylation of antigens, ithas not, to date, been possible to prepare antigens derived from theO-glycosylated proteins of these pathogenic microorganisms, which caneffectively be used in an immunogenic or immunization composition and/orin a diagnostic test.

Specifically:

-   -   the active proteins which represent only a small percentage of        the proteins produced by these microorganisms are purified with        very low yields, using methods which are dangerous due to the        handling of large amounts of these pathogenic agents,    -   the proteins, produced in heterologous expression systems        (eukaryotic cells or bacteria incapable of glycosylating), have        a low antigenic activity,    -   the proteins produced in homologous expression systems such        as M. smegmatis have an acceptable antigenic activity but they        are produced in insufficient amounts using complex methods.

Consequently, the inventors have set themselves the aim of preparingimmunodominant antigens capable of inducing a protective humoral and/orcellular immune response, which, on the one hand, when administeredalone or in combination with other antigens, may constitute a vaccinewhich can be used in all individuals, including immunodepressedindividuals (disappearance of the risk linked to the use of a livevaccine) and, on the other hand, may be used for diagnostic purposes.

They have found that certain glycopeptides derived from pathogenicmicroorganisms which synthesize glycoproteins (and in particularmycobacteria) exhibit an antigenic activity which is at least equal, ifnot greater than, that of the deglycosylated native protein or of therecombinant protein produced in E. coli.

It is also an aim of the invention to develop means, which are simple toimplement, for producing these glycopeptides in large amounts.

A subject of the present invention is immunogenic glycopeptides selectedfrom the group consisting of:

a₁) glycopeptides essentially consisting of a glycosylated T epitope,comprising from 14 to 25 amino acids, among which at least one neutralamino acid is bonded to a disaccharide or to a trisaccharide (glycosidicbond) and at least 15% of said amino acids are prolines, one of theprolines being located in position −1 to −4, relative to the position ofsaid neutral amino acid, which glycopeptides, derived from a pathogenicmicroorganism, are:

-   -   presented by a class II MHC molecule,    -   specifically recognized by CD4+ T lymphocytes induced by        immunization with the native glycoprotein from which they are        derived, but are not recognized by the CD4+ T lymphocytes        induced by immunization with a non-glycosylated peptide with the        same sequence and    -   capable of inducing a proliferation of said CD4+ T lymphocytes        which recognize them and the secretion of cytokines by said        lymphocytes, and

b₁) glycopeptides which have a sequence of 15 to 39 amino acidsincluding the sequence of the glycopeptide as defined in a₁), excludingthe glycopeptide of sequence SEQ ID NO:11, derived from the Apa which isdescribed by Dobos et al. (J. Bacteriol., 1996, 178, 2498-2506).

These glycopeptides consisting essentially of a glycosylated T epitopeare recognized by CD4+ T lymphocytes via this glycosylated T epitope.Specifically, after immunization with live bacilli of the tuberculosisgroup, there are many more T lymphocytes specific for theseglycopeptides than T lymphocytes specific for the non-glycosylatedpeptides with the same sequence.

Advantageously, said glycopeptides have an antigenic activity which isat least 10 times greater, preferably at least 30 times greater, thanthat of a control peptide with the same sequence.

Said glycopeptides have the following advantages:

-   -   induction of a protective cellular-type immune response and        possibly of a humoral response, and possible use as antigens in        immunodepressed individuals,    -   antigenic activity at least equal to, if not greater than,        conventional antigens (culture of said attenuated live        microorganisms, mixtures of antigens prepared from said cultures        or non-glycosylated peptides) since they are recognized by a        greater number of T lymphocytes specific for the pathogenic        microorganism,    -   very narrow specificity, which makes it possible both to        eliminate the problems of cross-reactivity with other        microorganisms, in particular with other a typical mycobacteria,        and to increase the effectiveness of the immunization and of the        diagnosis of the pathogenic microorganisms; specifically, they        are more specific given that their oligosaccharide residues,        which are present exclusively in said pathogenic microorganisms,        contribute in an essential manner to the definition of the T        epitope recognized by the CD4+ T lymphocytes; they thus        constitute specific antigens for immunization and for diagnosing        infections with pathogenic organisms capable of O-glycosylating        some of their proteins (bacilli of the tuberculosis complex,        Flavobacterium meningosepticum, Candida albicans, etc.),    -   use in immunodepressed individuals since they are totally        apathogenic,    -   production possible in large amounts,    -   better standardization of active doses and of the effectiveness        of the vaccine,    -   ease of storage and of use.

According to an advantageous embodiment of said glycopeptides, saidneutral amino acids are selected from the group consisting of serine andthreonine.

According to an advantageous arrangement of this embodiment of saidglycopeptides, they contain from 1 to 7 threonine residues bonded to adisaccharide or to a trisaccharide.

According to another advantageous embodiment of said glycopeptides, saiddisaccharide or trisaccharide is a dimer or a trimer of hexose,preferably a mannose.

According to yet another advantageous embodiment of said glycopeptides,said glycosidic bond is an α-(1,2) bond.

According to yet another advantageous embodiment, said glycopeptides arederived from a pathogenic microorganism capable of O-glycosylatingproteins, preferably Mycobacterium tuberculosis or Candida albicans.

In accordance with the invention, said glycopeptides are preferablyderived:

-   -   from the Apa protein of M. tuberculosis (Genbank number X80268)        or    -   from the Rv1796 protein encoded by the Rv 1796 gene, with        reference to the annotation of the sequence of the genome of M.        tuberculosis strain H37Rv (Sanger bank).

Preferably, said glycopeptide is selected from the group consisting of:

-   -   a 39 amino acid glycopeptide, the sequence (SEQ ID NO:1) of        which is that which extends from positions 1 to 39 of the        sequence of the Apa protein and in which at least one of the        threonine residues in position 10, 18 and 27 of SEQ ID NO:1 is        bonded to a disaccharide or trisaccharide via a glycosidic bond,    -   a 26 amino acid glycopeptide, the sequence (SEQ ID NO:2) of        which is that which extends from positions 261 to 286 of the        sequence of the Apa protein (C-terminal sequence) and in which        the threonine residue in position 17 of SEQ ID NO:2 is bonded to        a disaccharide or trisaccharide via a glycosidic bond, and    -   a 35 amino acid glycopeptide, the sequence (SEQ ID NO:3) of        which is that which extends from positions 169 to 203 of the        sequence of the Rv 1796 protein and in which at least one of the        threonine residues in position 4, 5, 7, 13, 15, 23 and 25 of SEQ        ID NO:3 is bonded to a disaccharide or trisaccharide via a        glycosidic bond.

A subject of the invention is also a method for synthesizing aglycopeptide as defined above, characterized in that it comprises thefollowing steps:

-   -   preparing, in solution, glycosylated neutral amino acids bonded        to a disaccharide or to a trisaccharide via a glycosidic bond,    -   synthesizing the glycopeptide, on a solid support, using the        amino acids required for producing the peptide sequence of said        glycopeptide and the glycosylated neutral amino acids obtained        above, and    -   cleaving the glycopeptide from the solid support.

According to an advantageous embodiment of said method, said neutralamino acid is selected from the group consisting of serine andthreonine.

According to an advantageous arrangement of this embodiment, when saidglycopeptides have the following sequences (T represents anO-glycosylated threonine functionalized with 2 or 3 glycosidic residues,and Ac represents an acetate function):

SEQ ID NO:1: H₂N-DPEPAPPVPTTAASPPSTAAAPPAPATPVAPPPPAAANT-CONH₂ SEQ IDNO:2: AcNH-PAPAPAPAGEVAPTPTTPTPQRTLPA-COOH: SEQ ID NO:3:AcNH-TIPTTETPPPPQTVTLSPVPPQTVTVIPAPPPEEG-CONH₂,said method comprises the following steps:

i) preparing, in solution, O-glycosylated threonines functionalized with2 or 3 glycosidic residues,

ii) synthesizing the peptides corresponding to the sequences SEQ IDNO:1, SEQ ID NO:2 and SEQ ID NO:3 mentioned above, on a solid support,using the amino acids required for producing these sequences and theO-glycosylated threonines obtained in step i),

iii) cleaving the peptides from the solid support, and

iv) introducing, by chemical synthesis, an amide function at theC-terminal end of the peptides SEQ ID NO:1 and SEQ ID NO:3, and anacetate function at the N-terminal end of the peptides SEQ ID NO:2 andSEQ ID NO:3.

The synthesis of the peptides SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3therefore corresponds to a conventional solid-phase peptide synthesisduring which glycosylated amino acids are introduced. As is known in thefield of solid-phase peptide synthesis, the amino acids used aresuitably protected and, if necessary, activated before beingincorporated one after the other into the peptide sequence. Similarly,the hydroxyls present on the glycosidic residues borne by the threoninesmust be suitably protected during the peptide synthesis.

Once the peptide synthesis has been carried out, the peptides areseparated from the solid support and deprotected. They can be purifiedby reverse-phase High Performance Liquid Chromatography.

According to an advantageous arrangement of this embodiment, theglycosidic residues borne by the O-glycosylated threonines prepared instep i) are hexoses, preferably mannoses, the mannose residuesadvantageously being bonded to one another via α-(1,2) bonds.

According to an advantageous mode of this arrangement, the threoninesfunctionalized with mannose residues are prepared as follows:

a₂) preparation of mannose derivatives of formulae (I) and (II):

in which P₁ and P₂, which may be identical or different, representgroups which protect a hydroxyl function, and X represents an activatedfunction, such as a bromine atom,

b₂) reaction of the derivative of formula (I) with the derivative offormula (II), then activation of the compound obtained, leading to theproduction of an activated derivative comprising two mannose residuesand corresponding to the formula (III):

in which P₁, P₂ and X are as defined in relation to formulae (I) and(II),

c₂) optionally, reaction of the compound of formula (III) with a mannosederivative of formula (I) as defined in a₂), then activation of thecompound obtained, leading to the production of an activated derivativecomprising three mannose residues and corresponding to the formula (IV):

in which P₁, P₂ and X are as defined in relation to formulae (I) and(II), and

d₂) condensation of the compound of formula (III) or of the compound offormula (IV) with a suitably protected threonine of formula (V):

in which P₃ represents a group which protects a primary amine functionand P₄ represents a group which protects a hydroxyl function,leading, respectively, to the production of a glycosylated threonine offormula (VI) or (VII):

in which P₁, P₂, P₃ and P₄ are as defined above.

The protective groups P₁, P₂, P₃ and P₄ may be chosen from thosedescribed in the work Protective Groups in Organic Synthesis, T. W.GREENE and P. G. M. WUTS, Second Edition, 1991, J. WILEY and Sons. Byway of examples and in a nonlimiting manner, P₁ and P₂ may representacetyl or benzoyl groups, P₃ may represent an Fmoc(9-fluorenylmethoxycarbonyl) group and P₄ may represent apentafluorophenyl group.

A subject of the present invention is also a method for selecting andscreening immunogenic glycopeptides using the peptide sequence of theproteins of a pathogenic microorganism, which may advantageously becarried out concomitantly with the method for synthesizing theglycopeptides in accordance with the invention, as defined above, whichmethod is characterized in that it comprises at least the followingsteps:

a₃) searching for and selecting, in and from the peptide sequence ofsaid proteins, at least one 14 to 25 amino acid sequence containing atleast one neutral amino acid bonded to a disaccharide or a trisaccharideand at least 15% of proline, one of the prolines being located inposition −1 to −4, relative to the position of said neutral amino acid,

b₃) preparing the glycopeptide(s) selected in step a₃), in accordancewith the method of synthesis defined above, and

c₃) selecting the glycopeptides the antigenic activity of which is atleast 10 times greater, preferably at least 30 times greater, than thatof a control peptide with the same sequence.

According to an advantageous embodiment of said screening method, priorto step a₃), it comprises a step for preselecting at least one antigenicglycoprotein.

According to another advantageous embodiment of said screening method,in step C₃), the antigenic activity of said glycopeptide is evaluated bymeasuring the activity of the CD4+ T lymphocytes of animals immunizedwith said attenuated pathogenic microorganism or with an antigenicfraction of said pathogenic microorganism.

The activation of the T lymphocytes can be demonstrated usingconventional immunology techniques, such as those described in Currentprotocols in Immunology (John E. Coligan, 2000, Wiley and son Inc,Library of Congress, USA). By way of example, mention may be made oflymphocyte proliferation assays, assays for the cytokines (protein ormRNA) synthesized by activated CD4+ T lymphocytes (immunoassay (ELISA)or polymerization chain reaction of the RT-PCR type) or, in the case ofM. tuberculosis, delayed-type hypersensitivity assays.

The present invention also encompasses the glycopeptides which can beobtained using the selection and screening method as defined above.

A subject of the present invention is also the use of at least oneglycopeptide in accordance with the invention or of a glycopeptide ofsequence SEQ ID NO:11, for preparing an immunogenic or immunizationcomposition or a diagnostic reagent.

The glycopeptides according to the invention which detect veryspecifically the cellular and/or humoral immunity induced by infectionwith a pathogenic microorganism, in particular M. tuberculosis, mayadvantageously be used for the diagnosis of tuberculosis by anytechnique which allows the detection of cellular immunity, thistechnique being known to those skilled in the art, per se. By way ofexample, mention may be made of T-lymphocyte proliferation assays andimmunoenzymatic assays for cytokines specific for CD4+ T lymphocytes, inparticular γ-IFN.

A subject of the present invention is also an immunogenic compositioncapable of inducing humoral and/or cellular immunity, characterized inthat it comprises at least one glycopeptide as defined above, combinedwith at least one pharmaceutically acceptable vehicle.

Because of the cooperation between CD4+ T lymphocytes and CD8+ Tlymphocytes or B lymphocytes in the setting up of a humoral or cellularimmune response, the glycopeptides of the invention may advantageouslybe used as a transport protein (carrier) for any other immunizationantigen in order to increase the effectiveness of the immunizationagainst said antigen. This antigen/carrier combination advantageouslymakes it possible to facilitate the selection and the amplification ofthe B and T lymphocytes specific for the immunization antigen.

A subject of the present invention is also an immunization compositionwhich is capable of inducing humoral and/or cellular immunity,characterized in that it comprises at least one glycopeptide as definedabove, combined with at least one pharmaceutically acceptable vehicleand, optionally, with at least one adjuvant.

According to an advantageous embodiment of said immunogenic orimmunization compositions, said glycopeptide is combined with a proteinor a protein fragment comprising at least one B epitope, one T epitopeof the CF4+type or one T epitope of the CD8+type.

For the purposes of the present invention, the terms “B epitope”, “Tepitope of the CD4+ type” and “T epitope of the CD8+ type”, relative tothe sequence of a protein, is intended to mean the fragment of thissequence which is capable of binding, respectively, to an antibody, to aT receptor of CD4+ lymphocytes and to a T receptor of CD8+ lymphocytes.

For the purposes of the present invention, the expression “combinationof the glycopeptide with a protein” is intended to mean both mixing andcoupling by any physical or chemical means, for example the expressionof a fusion between the sequence of the glycopeptide and that of theprotein or of the protein fragment.

The adjuvants used are conventionally used adjuvants; advantageously,they are chosen from the group consisting of aluminium hydroxide andsqualene.

Said glycopeptide may optionally be combined with any other means, knownper se to those skilled in the art, which makes it possible to increasethe immunogenicity of a peptide. By way of example, mention may be madeof coupling to a carrier peptide, which enables the production of abranched multimerized peptide, such as that described by Wilkinson etal., 1999, Eur. J. Immunol., 29, 2788-2796.

A subject of the present invention is also antibodies, characterized inthat they are directed against one or more of the glycopeptidesaccording to the present invention.

According to an advantageous embodiment of said antibodies, they areselected from monoclonal antibodies and polyclonal antibodies.

A subject of the present invention is also a diagnostic reagent,characterized in that it is selected from the group consisting of theglycopeptides and the antibodies according to the invention.

A subject of the present invention is also a method for detecting aninfection with a pathogenic microorganism, characterized in that itcomprises bringing a biological sample from a patient likely to beinfected with said pathogenic microorganism into contact with adiagnostic reagent as defined above (antibodies or glycopeptides,depending on the case) and detecting the formation of anantibody/microorganism present in the biological sample complex or aglycopeptide(s)/antibodies present in the sample complex.

Besides the arrangements above, the invention comprises even morearrangements, which will emerge from the following description whichrefers to examples of implementation of the present invention and alsoto the attached diagrams in which:

FIG. 1 illustrates the measurement, using a delayed-typehypersensitivity assay, of the antigenic activity of the native Apapurified from M. tuberculosis, as a function of the kinetics ofdigestion of the Apa protein by α-mannosidase. The results are expressedin tuberculin units per mg of protein as a function of time in hours,

FIGS. 2.1 and 2.2 illustrate the mass spectrometry analysis of themannose composition of the Apa molecules, as a function of the kineticsof digestion of the Apa protein with α-mannosidase (FIG. 2.1 showsanalysis at 0 minutes of digestion, 30 minutes of digestion, 60 minutesof digestion, and 4 hours of digestion; FIG. 2.2 shows analysis at 16,24, 48 and 72 hours of digestion). The number of mannose residuescorresponding to each peak of the Apa protein is indicated and theoverall antigenic activity of the product of the Apa digestion isindicated at the various times studied,

FIG. 3 illustrates the measurement, using a delayed-typehypersensitivity assay, of the antigenic activity of a glycopeptide,termed Lip, derived from the Rv 1796 protein (SEQ ID NO:3). The standardpurified proteins from M. tuberculosis (PPD) are used as a positivecontrol at the dose of 0.25 μg in 0.1 ml. The Lip peptide is used at thedose of 0.02 μg in 0.1 ml. The Lip peptides treated with α-mannosidaseor subtilisin are negative at the same doses. The results are expressedby the erythema reaction diameter,

FIG. 4 illustrates the antigenic activity of the Lip peptide using an invitro lymphocyte proliferation assay. The recognition of theglycosylated Lip peptide (native Lip) by the T lymphocytes is comparedto that of the deglycosylated peptide (Lip+α-mannosidase) or of the Lippeptide combined with an anti-T-lymphocyte CD4+ receptor antibody (Lip+anti Cd4),

FIG. 5 illustrates the measurement, using a delayed-typehypersensitivity assay, of the antigenic activity of the native Apapurified from M. tuberculosis (native Apa) or of the deglycosylatedrecombinant Apa produced in E. coli (E. coli rApa), as a function of theimmunization of guinea pigs. The latter were immunized beforehand withlive BCG injected intradermally or with the plasmids pAG831 or pAG832,containing the coding sequence of Apa, placed under the control of thecytomegalovirus early promoter. The immunization of the guinea pigs withthe plasmids produces a sensitization which can be revealed by adelayed-type hypersensitivity reaction. The two types of antigen areequivalent for engendering this reaction, whereas, after an immunizationwith BCG, only the glycosylated native Apa is antigenic,

FIG. 6 represents the preparation of units comprising two or threemannose residues bonded via α-(1,2) bonds, and

FIGS. 7 (7 a and 7 b) represents the preparation of threoninesfunctionalized with two or three mannose units.

EXAMPLE 1 Importance of the Number of Oligosaccharide Residues in theAntigenicity of the Apa Protein

1. Materials and Methods

a) Limited Deglycosylation of Apa by Digestion with α-mannosidase

450 μg of Apa protein purified from the culture supernatant of M.tuberculosis, according to the protocol described by Horn et al.,mentioned above, are diluted in a 450 μl volume of buffer A (100 mMCH₃COO⁻Na⁺, 2 mM ZnCl₂).

At the initial timepoint, 75 μl of the Apa protein solution are removed,diluted in 25 μl of buffer A and frozen as a control. 125 μl ofα-mannosidase at 1 mg/ml (3 IU/ml, Oxford Glycosciences) are then addedto the 375 μl of the Apa solution and the 500 μl reaction volume isincubated at 37° C. After 30 min, 1 h, 4 h, 16 h and 24 h, 100 μl of thereaction are removed and frozen at −20° C.

b) Purification of the Digestion Products

The 100-μl samples are heated for 2 min at 90° C. and are then abruptlycooled, dried under vacuum and resuspended in 300 μl of trifluoroaceticacid at 0.1% in water (solution B).

The Apa digestion products are separated from the α-mannosidase on areverse-phase chromatography column (Ressource RPC, Pharmacia), using agradient of 0 to 90% acetonitrile in solution B, in 90 min. The Apa iseluted from the column at the time t=68 min, corresponding to 51.5%±0.5%of acetonitrile. The fractions corresponding to the Apa are collected,lyophilized, resuspended in a solution of butanol at 5% in water(solution C) and then dried under vacuum. The purified samples are thenresuspended in 100 μl of solution C.

c) Biochemical Analysis of the Apa Digestion Products

The oligosaccharide composition of each sample is analyzed by massspectrometry under the conditions described in Horn et al., mentionedabove.

The absorption at 210 nm is measured in order to evaluate the relativeamount of protein present in each sample.

Next, the samples are dried and their concentration is adjusted to 1mg/ml in a titration buffer (buffer D: PBS, 0.9% NaCl, 0.05% Tween 80).

d) Biological Titration of the Antigenic Activity of the Products ofLimited Digestion of Apa with α-Mannosidase, in a Delayed-TypeHypersensitivity Assay

The antigenic activity is measured using a delayed-type hypersensitivityassay on guinea pigs immunized 3 months beforehand by an intradermalinjection of 2 mg of live BCG at 2 injection points.

Each sample is diluted to a concentration of 2 μg/ml in buffer D and 100μl of this dilution (0.2 μg) are injected intradermally into batches of2 previously immunized guinea pigs.

The various batches of animals are as follows:

-   -   batch 1: negative control having received 100 μl of buffer D    -   batch 2: Apa t=0    -   batch 3: Apa t=30 min    -   batch 4: Apa t=1 h    -   batch 5: Apa t=4 h    -   batch 6: Apa t=16 h    -   batch 7: Apa t=24 h    -   batch 8: positive control (0.25 μg of standard purified proteins        from Mycobacterium tuberculosis (PPD) corresponding to 10        tuberculin units (TU).

24 h after the injection, the mean of the erythema reaction diameter ismeasured for the various batches of animals and the tuberculin titre ofthe samples is determined with respect to the PPD standard.

2. Results

The results are illustrated by FIGS. 1 and 2.

The analysis of the antigenic activity of the Apa as a function of thekinetics of digestion with α-mannosidase (FIG. 1) shows that theantigenic activity of the Apa is gradually lost during the digestionwith α-mannosidase: 66% in 1 h, 86% in 4 h and 97 to 99% for the longerdigestions.

The analysis of the mannose composition of the products obtained at thevarious digestion times (FIG. 2) shows that:

-   -   the native Apa molecules have 6 to 8 mannose residues, and    -   the Apa molecules on which there remain 3 to 6 mannose residues        lose 86% of their antigenic activity.

It has been shown that the oligomannose composition of Apa is asfollows: a dimannose (T₁₀ and T₁₈), a mannose (T₂₇), a mannose, adimannose or a trimannose (T₂₇₇), Dobos et al., mentioned above. Inaddition, α-mannosidase is an exomannosidase.

Consequently, the results indicate that:

-   -   the loss of 1 or 2 of the terminal mannoses of the 4        oligomannose chains of Apa causes a drastic loss of the        antigenic activity, and    -   the antigenicity of Apa is linked to the presence of a dimannose        or of a trimannose on one or more of the glycosylated threonine        residues.

EXAMPLE 2 Demonstration of the Lip glycopeptide of M. tuberculosis.

1. Materials and Methods

a) Purification of the Glycopeptide

a1) Preparation of the Crude Material

Bacteria of the Mycobacterium tuberculosis (H37Rv) strain are culturedfor 20 days on a Sauton synthetic medium (culture medium, H. Cassagne,1961, Ed. Institut Pasteur, volume 2, page 242). The molecules secretedinto the medium are concentrated on an ultrafiltration membrane (PM10,AMICON) in such a way as to retain only the molecules of molecular massgreater than 10 000 Da, and then they are lyophilized. Approximately 10g of lyophilisate are obtained for 60 litres of culture medium.

a2) Molecular Filtration (step 1)

A preparative column is filled with Sup75 prepgrade matrix, Pharmacia.This 50×750 mm column is equilibrated with a phosphate buffer (50 mMNa₂/K PO₄, pH 7.1; 100 mM NaCl; 4% butanol) at a flow rate of 1 ml/min.The crude material above is taken up in the equilibration buffer at afinal concentration of 10 g per 100 ml and clarified by centrifugationat 43 000 g for 4 h, then by filtration over a 0.22 μm filter.Injections of 13 ml are performed and the various fractions detected viatheir absorbence at 280 nm are concentrated on a PM10 membrane and thenlyophilized.

The fraction eluted between 700 and 800 ml is very antigenic:delayed-type hypersensitivity is observed in guinea pigs immunized withlive BCG; this fraction is, on the other hand, relatively inactive inguinea pigs immunized with heat-inactivated BCG.

a3) Ion Exchange (step 2)

A 24×250 mm Pharmacia Source 15Q preparative column (15 μm) isequilibrated with a 20 mM tris/HCl, pH 8, 4% butanol buffer at a flowrate of 5 ml/min with a maximum pressure of 8 bar. A linear NaClgradient of 0 to 150 mM in the same buffer is applied after injecting500 mg of the fraction above dissolved in 10 ml of initial buffer. Thefractions eluted are detected by absorption at 280 nm, concentrated on aPM10 membrane and then lyophilized.

The fraction eluted between 40 and 75 mM NaCl is very antigenic;delayed-type hypersensitivity is observed in guinea pigs immunized withlive BCG; this fraction is, on the other hand, relatively inactive inguinea pigs immunized with heat-inactivated BCG.

a4) Reverse Phase on a C8 Column (step 3)

A 4.6×100 mm Pharmacia RPC column (Reversed Phase Column) Resource 15RPCis equilibrated with a 20 mM CH₃COO⁻NH₄ ⁺ buffer, pH 6.5, at a flow rateof 1 ml/min. A nonlinear acetonitrile gradient, of between 0 and 90%, isapplied after injecting onto the column 10 mg of the fraction above, in2 ml of buffer. The fractions eluted are detected at 280 nm and thenconcentrated under vacuum at 40° C. before being lyophilized.

The fraction eluted between the acetonitrile concentrations of 18 and22% is very antigenic in terms of revealing delayed-typehypersensitivity in guinea pigs immunized with live BCG and relativelyinactive in guinea pigs immunized with heat-inactivated BCG.

a5) Reverse Phase on a C18 Column (step 4)

A C18 reverse-phase microbore column (Browlec lab. 1×250 mm) isequilibrated with a 20 mM CH₃COO³¹ NH₄ ⁺ buffer, pH 6.5, at a flow rateof 1 ml/min. A nonlinear acetonitrile gradient of 0 to 90% is appliedafter injecting the fraction above onto the column.

A fraction detected only at 220 nm is eluted with a concentration ofapproximately 11% of acetonitrile. This fraction (3 mg) is very activein terms of revealing delayed-type hypersensitivity reactions in guineapigs immunized with live bacteria and relatively inactive in guinea pigsimmunized with heat-inactivated BCG.

b) Biochemical Analysis of the Purified Glycopeptide

The fraction obtained in the final purification step was sequenced usinga modified Edman technique (Applied Biosystems 473A), according to themanufacturer's instructions.

The composition of each sample is analyzed by mass spectrometry(MALDI-TOF spectrometer) under the conditions described by Horn et al.,mentioned above.

c) Digestion of the Glycopeptide with α-Mannosidase

9 μg of the glycopeptide purified above are dissolved in 65 μg of 100 mMCH₃COO⁻Na⁺ buffer, pH 5, and then 3 μl of a 1 mg/ml α-mannosidasesolution (Oxford Glyco System), i.e. 90 mU of α-mannosidase, are added.The reaction is incubated for 24 h at 37° C. so as to obtain totaldigestion, and then the product obtained is dried under vacuum.

d) Digestion of the Peptide with Subtilisin

690 ng of the glycopeptide purified above are dissolved in 5 μl of 100mM ammonium carbonate buffer, pH 8, and then 1 μl of a 100 μg/mlsubtilisin solution, i.e. approximately 100 ng, is added. The reactionis incubated for 24 h at 37° C. and then the reaction product is driedunder vacuum and taken up in the titration buffer (buffer D).

e) Biological Titration of the Antigenic Activity of the GlycopeptideUsing a Delayed-Type Hypersensitivity Assay

0.02 μg of the glycopeptide purified above, nondigested or digested withα-mannosidase or subtilisin, are injected in batches of previouslyimmunized guinea pigs, according to the protocol described in Example 1.The results are expressed by the value of the erythema reactiondiameter. The control consists of 0.25 μg of PPD, corresponding to 10TU.

f) Measurement of the Antigenic Activity of the Glycopeptide Using an InVitro Lymphocyte Proliferation Assay

The conditions of the assay are those described in Horn et al.,mentioned above.

2. Results

a) Purification and Biochemical Analysis of the Lip Glycopeptide

The mass measurement performed on the purified glycopeptide indicatesthe presence of complex molecules probably glycosylated with mannoses,given the presence of measurements which differ by a value of 162 massunits. A mass of 6 951 Da, which corresponds to the mass of the peptidetreated with α-mannosidase, is taken as the minimum mass of thesemolecules.

The N-terminal sequence of the purified glycopeptide indicates thepresence of a major sequence TIPTT . . . (amino acids 1-5 of SEQ IDNO:3) and of a minor sequence IPTTE . . . . (amino acids 2-6 of SEQ IDNO:3).

These results are compatible with a mannosylated glycopeptide, termedLip, the sequence (SEQ ID NO:3) of which is that of an N-terminalfragment of a peptide derived from the protein encoded by the Rv1796gene of M. tuberculosis, which extends from positions 169 to 239 of saidprotein, with reference to the annotation of the sequence of the genomeof M. tuberculosis strain H37Rv from the Sanger bank.

b) Measurement of the Antigenic Activity of the Lip Glycopeptide Using aDelayed-Type Hypersensitivity Assay

The glycopeptide is very active in terms of revealing delayed-typehypersensitivity reactions in guinea pigs immunized with live bacteria,on the other hand it is relatively inactive in guinea pigs immunizedwith heat-inactivated BCG.

The antigenic activity of the glycopeptide increases during thepurification steps:

-   -   Step 1: The fraction obtained has an activity of 180 000 TU/mg        in guinea pigs immunized with live BCG and of 10 000 TU/mg in        guinea pigs immunized with heat-inactivated BCG.    -   Step 2: The fraction obtained has an activity of 900 000 TU/mg        in guinea pigs immunized with live BCG and of 30 000 TU/mg in        guinea pigs immunized with heat-inactivated BCG.    -   Step 3: The purified fraction has an activity of greater than 1        000 000 TU/mg in guinea pigs immunized with live BCG and of less        than 30 000 TU/mg in guinea pigs immunized with heat-inactivated        BCG.

The results illustrated in FIG. 3 show that:

-   -   the action of α-mannosidase for 24 h at 37° C. causes a loss of        more than 95% of the antigenic activity: the fraction dropped        from an activity of 1 000 000 TU/mg to an activity of less than        30 000 TU/mg after deglycosylation,    -   the action of subtilisin abolishes the antigenic activity, and    -   at an equivalent amount of proteins, the Lip glycopeptide is at        least 10 times more active than the standard purified proteins        from Mycobacterium tuberculosis (PPD).        c) Measurement of the Antigenic Activity of the Lip Glycopeptide        Using an In Vitro Lymphocyte Proliferation Assay

The results illustrated in FIG. 4 show that the T-lymphocyteproliferation is dependent on the peptide concentration. Thisproliferation is marginal when the T lymphocytes are treated with anantibody directed against CD4 molecules or when the glycopeptide istreated with α-mannosidase.

EXAMPLE 3 Demonstration of the Role of the Oligosaccharide Residues ofApa in Defining T Epitopes, by Immunization with Naked DNA Encoding theApa Protein

1. Materials and Methods

a) Construction of a Plasmid Containing the Sequence Encoding the ApaProtein

The plasmid pS65T (Clontech) containing the sequence of thecytomegalovirus early promoter is cleaved with the NheI and BspEIrestriction enzymes, repaired with the Klenow enzyme and then ligated soas to obtain the plasmid pAG800.

The plasmid pAG800 is cleaved with the ApaI enzyme and ligated with theoligonucleotide 12M48 (5′CAACGTTGGGCC 3′; SEQ ID NO:4) hybridized toitself, so as to give the plasmid pAG802.

An 875 base pair fragment containing the coding sequence of Apa lackingthe signal sequence is amplified, by polymerase chain reaction (PCR),from the plasmid pLA34-2 (Laqueyrerie, 1995, Infect. Immun., 63,4003-4010), using:

-   -   the oligonucleotides 22M42 (5′ TCCCAAGCTTTTGGTAGCCG 3′; SEQ ID        NO:5) and 33M44 (5′ CTAGGATCCACCATGCCGGAGCCAGCGCCCCCG 3′; SEQ ID        NO:6).

The oligonucleotide 33M44 was synthesized in such a way as to contain aconsensus translation initiation site of the Kozak type (Nucl. AcidsRes., 1987, 15, 8125-8148). The fragment obtained by PCR is cleaved withBamHI and EcoRV and inserted into the plasmid pAG802 cleaved with BgIIIand SmaI, so as to give the plasmid pAG803. During these operations, theoligonucleotide sequence 5′CAACGTTGGGCC 3′ (SEQ ID NO:4) is lost; thissequence, termed Psp1046 ISS, is considered to be an immunostimulantsequence which increases immune responses in the same way as thesequence IL-12p40 ISS (Lipford GB et al., 1997, Eur. J. Immunol., 27,3420-3426).

A Psp1046 ISS sequence is inserted at the BamHI site of the plasmidpAG803 by cloning the oligonucleotide 25M45 (5′GATCCGGGGGGGAACGTTGGGGGGG 3′; SEQ ID NO:7) hybridized with theoligonucleotide 25M46 (5′ GATCCCCCCCCAACGTTCCCCCCCG 3′; SEQ ID NO:8), soas to obtain the plasmid pAG831.

An IL-12p40 ISS sequence is inserted at the BamHI site of the plasmidpAG803 by cloning the oligonucleotide 24M63 (5′ AGCGCTATGACGTTCCAAGGGCCC3′; SEQ ID NO:9) hybridized with the oligonucleotide 24M64 (5′GGGCCCTTGGAACGTCATAGCGCT 3′; SEQ ID NO:10), so as to obtain the plasmidpAG832.

After transforming Escherichia coli strain XLl Blue, the plasmids aboveare amplified in LB culture medium (Sambrook et al., Molecular cloning:A laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989) containing 25 μg/ml of kanamycin. After a prior stepfor eliminating the endotoxin by treating the bacterial lysates withTriton X-114 (1%), the plasmid DNA is purified on MaxiPrep QIA filtercolumns (QIAGEN) according to the manufacturer's indications.

b) Immunization of Guinea Pigs with the Plasmids pAG831 and pAG832

Guinea pigs (Hartley) weighing 300 to 400 g are immunized with 50 μg ofthe DNA of the plasmids pAG831 or pAG832, prepared and purified asindicated above, by giving 2 intradermal injections into the flanks.

The control consists of a group of guinea pigs immunized with live BCGunder the conditions described in Example 1 or in Example 2.

c) Measurement of the Antigenic Activity of the Apa Protein Produced byEukaryotic Cells in Guinea Pigs Immunized with Naked DNA, Using aDelayed-Type Hypersensitivity Assay

One and two months after immunization, the delayed-type hypersensitivityreactions are measured with respect to the native Apa protein or to therecombinant Apa protein produced in a transformed strain of Escherichiacoli, which proteins are purified according to the protocol described inHorn et al., mentioned above.

The native Apa and the recombinant Apa are injected intradermally at thedose of 0.2 μg in 100 μl of titration buffer (buffer D). The antigenicactivity is measured as described in Example 2.

2. Results

The results illustrated by FIG. 5 are as follows:

-   -   The guinea pigs immunized with the plasmid pAG831 or pAG832        containing the coding sequence of Apa under the control of a        eukaryotic promoter develop, in the vast majority of cases, an        immune response directed against the native Apa protein        (antibodies and a T-response of the CD4+ type which can be        measured using a delayed-type hypersensitivity assay or an in        vitro T-lymphocyte proliferation assay when they are brought        together with the antigens). In the animals corresponding to the        native Apa antigen, the CD4+ T lymphocyte responses against the        antigen deglycosylated via the enzymatic pathway or against the        non-glycosylated recombinant antigen originating from E. coli        are of the same strength as the responses observed with the        glycosylated native antigen.    -   On the other hand, the guinea pigs immunized with live BCG show        a delayed-type hypersensitivity reaction only in response to the        native Apa. These animals develop no reaction or develop a        greatly decreased reaction in response to the non-glycosylated        recombinant Apa produced in E. coli as indicated above.

These results provide the following teachings:

1) The results observed in the animals immunized with naked DNA encodingApa indicate that the capacity of the Apa protein to be phagocytosed andpresented by macrophages or dendritic cells is identical for the nativeor recombinant (non-glycosylated) Apa protein.

2) The combination of the results above with the results observed in theanimals immunized with live BCG indicate that the absence of response tothe deglycosylated Apa protein is not due to a decrease in its capacityto be presented by macrophages or dendritic cells, but to an absence ofrecognition by CD4+ T lymphocytes. Consequently, the oligomannoseresidues of the side chains of the Apa or Lip proteins in the nativeform, such as those produced by M. tuberculosis or by live BCG, play arole in the constitution of T epitopes recognized by CD4+ T lymphocytes.

EXAMPLE 4 Preparation of the Glycosylated Peptides SEQ ID NO:1, SEQ IDNO:2 and SEQ ID NO:3

1) Preparation of the Glycosylated Synthons 15, 16 and 19

Prior to the peptide synthesis, glycosylated synthons, i.e. threoninesfunctionalized with two or three mannose residues, are prepared.

Preparation of the Compounds 5 and 8 (FIG. 6)

The preparation of the compounds 5 and 8 is described by H. FRANZYK etal. in J. Chem. Soc. Perkin Trans. 1, 1995, 2883-2898 and by R. K. NESSet al., in J. Am. Chem. Soc. Perkin, 1950, 72, 2200-2205, respectively.

The commercial peracetylated mannose 1 (i.e.1,2,3,4,5-penta-O-acetyl-α-D-mannopyranose) is brominated in theanomeric position by the action of hydrogen bromide in acetic acid, asdescribed by A. LEVENE et al. in J. Biol. Chem., 1931, 90, 89-98. Theactivated intermediate 2 is cyclized to the orthoester 3 in a2,6-dimethylpyridine/methanol mixture. The regioselective opening of theorthoester by acid hydrolysis at 0° C. in a 10% aqueous trifluoroaceticacid/acetonitrile mixture produces1,3,4,6-tetra-O-acetyl-β-D-mannopyranose (5). The regioisomer 4 is alsoisolated.

The commercial mannose 6 is perbenzoylated to 7 by the action of benzoylchloride in pyridine. The latter is activated to 8 by the action ofhydrogen bromide in acetic acid. In this protocol and in those whichfollow, activation methods other than by the action of hydrogen bromidemay, however, be used, such as they are known to those skilled in theart.

Preparation of the Disaccharides 10 and 12 (FIG. 7 a)

The preparation of the compounds 10 and 12 is described by A. JANSSON etal. in J. Chem. Soc. Perkin Trans. 1, 1992, 1699-1707 and by H. FRANZYKet al. (ibid), respectively. The compounds 2 and 5 are condensed in thepresence of silver trifluoromethanesulphonate (or any other condensationreaction promoter) in dichloromethane so as to produce the peracetylateddisaccharide 9, which is then activated to the brominated precursor 10by the action of hydrogen bromide in acetic acid. According to anidentical protocol, the compounds 5 and 8 are condensed to give thecompound 11, itself activated to 12.

Preparation of the Trisaccharide 18 (FIG. 7 a)

The activated disaccharide 12 is condensed onto the monosaccharideacceptor 5, in the presence of silver trifluoromethanesulphonate indichloromethane, so as to produce the peracetylated trisaccharide 17,which is then activated to the brominated precursor 18 by the action ofhydrogen bromide in acetic acid.

Preparation of the Synthons 15 and 16, Carrying Two Mannose Units, andof the Synthon 19, Carrying Three Mannose Units (FIG. 7 b)

As described by I. SCHON et al. in Synthesis, 1986, 303-305, the acidfunction of the commercial threonine 13, the primary amine function ofwhich is protected by an Fmoc group, is blocked in the form of an esterby the action of pentafluorophenol (pfp) in the presence ofdicyclohexylcarbodiimide (DCCI) so as to produce the acceptor precursor14.

The preparation of the synthons 15 and 16 is described by A. JANSSON etal. (ibid) and by H. FRANZYK et al. (ibid), respectively. Thecondensation of the compound 14 with the activated disaccharides 10 and12, carried out in the presence of silver trifluoromethane-sulphonate indichloromethane, produces the synthons 15 and 16, respectively.According to the same protocol, the condensation of the compound 14 withthe activated trisaccharide 18 produces the synthon 19.

2) Preparation of the Glycosylated Peptides SEQ ID NO:1, SEQ ID NO:2 andSEQ ID NO:3

The peptides are synthesized in solid phase using Fmoc chemistry. Thepeptide synthesis is performed on an automatic synthesizer, using theamino acids required for producing the desired sequences, whileincorporating the glycosylated synthons, which are in the form ofactivated esters of pentafluorophenol (synthons 15, 16 and 19).

Depending on the synthons used, either peptides comprising threoninesfunctionalized with two mannose residues (incorporation of the synthons15 and/or 16 during the peptide synthesis) or peptides comprisingthreonines functionalized with three mannose residues (incorporation ofthe synthon 19 during the peptide synthesis) or peptides comprising boththreonines functionalized with two mannose residues and threoninesfunctionalized with three mannose residues (incorporation of thesynthons 19 and 15 and/or 16 during the peptide synthesis) are obtained.

At the end of synthesis, after cleavage of the peptides from the solidsupport using trifluoroacetic acid and deprotection of the various aminoacids and of the hydroxyl functions of the mannoses, the peptides arepurified by reverse-phase High Performance Liquid Chromatography (HPLC).Their structure is controlled using techniques known to those skilled inthe art, such as mass spectrometry and amino acid analysis.

The amide functions (in the C-terminal position of the peptides SEQ IDNO:1 and SEQ ID NO:3, and acetate functions (in the N-terminal positionof the peptides SEQ ID NO:2 and SEQ ID NO:3) are then introduced bychemical synthesis, using organic chemistry techniques known to thoseskilled in the art.

EXAMPLE 5 Demonstration of the Role of the Oligosaccharide Residuals ofApa in Defining T Epitopes, by Immunization with an Apa Peptide Producedin E. coli

1) Materials and Methods

A peptide corresponding to positions 250 to 280 of Apa was produced inE. coli, in the form of a fusion with a fragment of Bordetella pertussiscyclase, according to the conventional techniques of cloning, expressionand purification of recombinant proteins in E. coli which are well knownto those skilled in the art (cf. for example, the protocols described inCurrent Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000,Wiley and Son Inc, Library of Congress, USA).

Three groups of 5 Hartley guinea pigs weighing 300 to 400 g wereimmunized, with 2 intradermal injections one month apart, with 20 μg ofthis purified Apa peptide, in 0.1 ml of an adjuvant solution.

Three groups of 4 guinea pigs immunized four months beforehand with liveBCG, under the conditions described in example 1, are used as controls.

One and two months after immunization, delayed hypersensitivityreactions were measured with respect to the native Apa protein, to therecombinant Apa protein produced in E. coli and to the deglycosylatedApa protein prepared as described in example 1, under the conditionsdefined in example 3.

2) Results

The delayed hypersensitivity reactions of the guinea pigs immunizedeither with the Apa fusion peptide or with the live BCG were measuredwith respect to the native Apa protein, to the recombinant Apa proteinproduced in E. coli and to the deglycosylated Apa protein. The resultsexpressed by the diameter of the erythema reaction (mm) are given intable I below:

TABLE I Antigenic activity of the Apa fusion peptide expressed in E.coli Antigen Live BCG Fusion peptide Native Apa 17-15-11-13 5-12-13-5-5E. Coli recombinant Apa 0 0 0 0 13-14-15-5-15 Deglycosylated Apa 0 0 0 0NT* *NT: not tested

As indicated in table I above, the delayed hypersensitivity reactionsobserved in the guinea pigs immunized with live BCG are considerableafter injection of native Apa molecules. The reactions are very weak orabsent after injection of the chemically deglycosylated molecules or ofthe molecules produced in E. coli. On the other hand, for the guineapigs immunized with the recombinant molecules corresponding to thefusion between the fragment of Bordetella pertussis cyclase and theinternal fragment of the Apa molecule, the sensitizations are identicalwith respect to the native or deglycosylated molecules.

These results show that the glycosylated T epitopes of the Apa moleculeare selectively recognized by the guinea pigs immunized with the livebacteria. They also show that the lack of, or reduced, recognition ofthe deglycosylated molecules by the guinea pigs is not associated with areduced intrinsic antigenicity of these molecules.

As emerges from the above, the invention is in no way limited to itsmethods of implementation, preparation and application which have justbeen described more explicitly; on the contrary, it encompasses all thevariants thereof which may occur to a person skilled in the art, withoutdeparting from the context or scope of the present invention.

1. Immunogenic glycopeptides selected from the group consisting of a₁)glycopeptides essentially consisting of a glycosylated T epitope,comprising from 14 to 25 amino acids, among which at least one neutralamino acid is bonded to a disaccharide or to a trisaccharide (glycosidicbond) and at least 15% of said amino acids are prolines, one of theprolines being located in position −1 to −4, relative to the position ofsaid neutral amino acid, which glycopeptides, obtained from a pathogenicmicroorganism, are: presented by a class II MHC molecule, specificallyrecognized by CD4+ T lymphocytes induced by immunization with the nativeglycoprotein from which they are obtained, but are not recognized by theCD4+ T lymphocytes induced by immunization with a non-glycosylatedpeptide with the same sequence and capable of inducing a proliferationof said CD4+ T lymphocytes which recognize them and the secretion ofcytokines by said lymphocytes, and b₁) glycopeptides which have asequence of 15 to 39 amino acids including the sequence of theglycopeptide as defined in a₁), excluding the glycopeptide of sequenceSEQ ID NO:11.
 2. The immunogenic glycopeptides according to claim 1,wherein said neutral amino acid is selected from the group consisting ofserine and threonine.
 3. The glycopeptides according to claim 1, whichcontain from 1 to 7 threonine residues bonded to a disaccharide or to atrisaccharide.
 4. The glycopeptides according to claim 1, wherein saiddisaccharide or trisaccharide is a dimer or a trimer of hexose.
 5. Theglycopeptides according to claim 4, wherein said hexose is a mannose. 6.The glycopeptides according to claim 1, wherein said glycosidic bond isan α-(1,2) bond.
 7. The glycopeptides according to claim 1, wherein saidpathogenic microorganism is capable of O-glycosylating proteins.
 8. Theglycopeptides according to claim 7, wherein said pathogenicmicroorganism is Mycobacterium tuberculosis or Candida albicans.
 9. Theglycopeptides according to claim 1, which are obtained from the Apaprotein of M. tuberculosis and encoded by the Rv1860 gene; or from theRv1796 protein of M. tuberculosis encoded by the Rv796 gene.
 10. Theglycopeptides according to claim 9, which are selected from the groupconsisting of: a 39 amino acid glycopeptide, the sequence (SEQ ID NO:1)of which is that which extends from positions 1 to 39 of the sequence ofthe Apa protein and in which at least one of the threonine residues inposition 10, 18 and 27 of SEQ ID NO:1 is bonded to a disaccharide ortrisaccharide via a glycosidic bond, a 26 amino acid glycopeptide, thesequence (SEQ ID NO :2) of which is that which extends from positions261 to 286 of the sequence of the Apa protein (C-terminal sequence) andin which the threonine residue in position 17 of SEQ ID NO:2 is bondedto a disaccharide or trisaccharide via a glycosidic bond, and a 35 aminoacid glycopeptide, the sequence (SEQ ID NO:3) of which is that whichextends from positions 169 to 203 of the sequence of the Rv 1796 proteinand in which at least one of the threonine residues in position 4, 5, 7,13, 15, 23 and 25 of SEQ ID NO:3 is bonded to a disaccharide ortrisaccharide via a glycosidic bond.
 11. A method for synthesizing aglycopeptide according to claim 1, comprising: preparing, in solution,glycosylated neutral amino acids bonded to a disaccharide or to atrisaccharide via a glycosidic bond, synthesizing the glycopeptide, on asolid support, using the amino acids required for producing the peptidesequence of said glycopeptide and the neutral amino acids obtainedabove, and cleaving the glycopeptide from the solid support.
 12. Themethod according to claim 11, wherein said neutral amino acid isselected from the group consisting of serine and threonine.
 13. Themethod according to claim 12, wherein, when said glycopeptides have thefollowing sequences (T represents an O-glycosylated threoninefunctionalized with 2 or 3 glycosidic residues, and Ac represents anacetate function): SEQ ID NO:1:H₂N-DPEPAPPVPTTAASPPSTAAAPPAPATPVAPPPPAAANT-CONH₂ SEQ ID NO:2:AcNH-PAPAPAPAGEVAPTPTTPTPQRTLPA-COOH: SEQ ID NO:3:AcNH-TIPTTETPPPPQTVTLSPVPPQTVTVIPAPPPEEG-CONH₂,

said method comprises: i) preparing, in solution, O-glycosylatedthreonines functionalized with 2 or 3 glycosidic residues, ii)synthesizing the peptides corresponding to the sequences SEQ ID NO:1,SEQ ID NO:2 and SEQ ID NO:3 mentioned above, on a solid support, usingthe amino acids required for producing these sequences and theO-glycosylated threonines obtained in step i), iii) cleaving thepeptides from the solid support, and iv) introducing, by chemicalsynthesis, an amide function at the C-terminal end of the peptides SEQID NO:1 and SEQ ID NO:3, and an acetate function at the N-terminal endof the peptides SEQ ID NO:2 and SEQ ID NO:3.
 14. The method according toclaim 13, wherein said glycosidic residues borne by the threonines arehexoses, preferably mannoses.
 15. The method according to claim 14,wherein the threonines functionalized with mannose residues are preparedusing the following steps: a₂) preparation of mannose derivatives offormulae (I) and (II):

in which P₁ and P₂, which may be identical or different, representgroups which protect a hydroxyl function, and X represents an activatedfunction, such as a bromine atom, b₂) reaction of the derivative offormula (I) with the derivative of formula (II), then activation of thecompound obtained, leading to the production of an activated derivativecomprising two mannose residues and corresponding to the formula (III):

in which P₁, P₂ and X are as defined in relation to formulae (I) and(II), c₂) optionally, reaction of the compound of formula (III) with amannose derivative of formula (I) as defined in a₂), then activation ofthe compound obtained, leading to the production of an activatedderivative comprising three mannose residues and corresponding to theformula (IV):

in which P₁, P₂ and X are as defined in relation to formulae (I) and(II), and d₂) condensation of the compound of formula (III) or of thecompound of formula (IV) with a suitably protected threonine of formula(V):

in which P₃ represents a group which protects a primary amine functionand P₄ represents a group which protects a hydroxyl function, leading,respectively, to the production of a glycosylated threonine of formula(VI) or (VII):

in which P₁, P₂, P₃ and P₄ are as defined above.
 16. A method forselecting and screening immunogenic glycopeptides using the peptidesequence of the proteins of a pathogenic microorganism, comprising: a₃)searching for and selecting, in and from the peptide sequence of saidproteins, at least one 14 to 25 amino acid sequence containing at leastone neutral amino acid bonded to a disaccharide or a trisaccharide andat least 15% of proline, one of the prolines being located in position−1 to −4, relative to the position of said amino acid, b₃) preparing theglycopeptide(s) selected in step a₃), in accordance with the methodaccording to claim 11, and c₃) selecting the glycopeptides the antigenicactivity of which is at least 10 times greater, preferably at least 30times greater, than that of a control peptide with the same sequence.17. The method according to claim 16, wherein, prior to step a₃), itcomprises a step for preselecting at least one antigenic glycoprotein.18. The method according to claim 16, wherein said neutral amino acid isselected from the group consisting of serine and threonine.
 19. Themethod according to claim 16, wherein, in step c₃), the antigenicactivity of said glycopeptide is evaluated by measuring the activity ofthe CD4+ T lymphocytes of animals immunized with said attenuatedpathogenic microorganism or with an antigenic fraction of saidpathogenic microorganism.
 20. A glycopeptide obtained using the methodaccording to claim
 11. 21. A composition capable of inducing humoraland/or cellular immunity, comprising at least one glycopeptide asclaimed in claim 1, a glycopeptide comprising SEQ ID NO: 11, or amixture thereof, combined with at least one pharmaceutically acceptablevehicle.
 22. A composition capable of inducing humoral and/or cellularimmunity, comprising at least one glycopeptide as claimed in claim 1,combined with at least one pharmaceutically acceptable vehicle and,optionally, with at least one adjuvant.
 23. The composition according toclaim 21, wherein said at least one glycopeptide is combined with aprotein or a protein fragment comprising at least one B epitope, one Tepitope of the CD4+ type or one T epitope of the CD8+0 type.
 24. Amethod of preparing the immunogenic composition as claimed in claim 21,comprising combining the at least one glycopeptide with at least onepharmaceutically acceptable vehicle.